Recent studies have indicated that ceramide, a sphingolipid contributes to the detrimental effects of different factors such as TNF-alpha, Fas L and endostatin to produce endothelial dysfunction. However, the mechanisms responsible for the production and actions of ceramide in endothelial cells are still poorly understood. Recently, a novel mechanism involving membrane lipid rafts and their aggregation has been reported to participate in ceramide-mediated transmembrane and intracellular signaling. In this proposal, we hypothesize that ceramide production and lipid raft clustering aggregate different signaling molecules such as NADPH oxidase subunits to form a membrane signaling amplification platform and thereby contributes to the reduction of NO bioavailability and endothelial dysfunction induced by different injury factors. We will first demonstrate the formation of lipid raft platforms and associated aggregation of the receptors in response to Fas L or TNF-alpha in bovine coronary arterial endothelial cells using fluorescent and confocal microscopy and flotation of detergent resistant membranes. We will also determine the association of ceramide with lipid raft clustering and explore the mechanisms of ceramide production by selective inhibition of related enzymes or by silencing the genes coding these enzymes such as sphingomyelinases using siRNA. Then, we will determine whether lipid raft clustering co-aggregates and activates endothelial NADPH oxidase by confocal microscopy, enzyme kinetic analysis and subunit translocation detection. Finally, we will determine whether lipid raft clustering stimulates 02- production and thereby contributes to endothelial dysfunction associated with NO using fluorescence imaging analysis of NO or 02- in the intact endothelium of bovine coronary arteries. The results of these studies will clarify the role of lipid raft clustering and ceramide production in mediating the actions of Fas L and TNF-alpha and provide new insights into the mechanisms contributing to endothelial dysfunction under different pathological conditions such as ischemia/reperfusion, atherosclerosis and hypertension.